This proposal seeks to define the intracellular effectors that account for the important role of phosphatidyl inositol-3 kinase (PI3K) signaling in B lymphocyte generation, function and maintenance. Activation of the PI3K signaling network recruits cytosolic effectors to the plasma membrane via pleckstrin homology (PH) domain-binding to PIP3,4,5. In particular, PDK1 has been identified as a pivotal downstream effector of PI3K that activates Akt, p70S6kinase, PKC enzymes and other targets that control cell metabolism, quiescence, proliferation and survival. Recently the Akt kinases have also been shown to negatively regulate the activity of the FOXO transcription factors. In doing so, expression of FOXO target genes is halted, leading to enhance cell cycle progression and survival. In B cells, we posit that the PDK1-dependent pathway acts in synergy with the PIP3,4,5-dependent Btk signaling pathway, which is crucial for NF-kappaB activation and Ca++ mobilization. To address this hypothesis, we will define the relative importance and distinct functions of the PDK1- and Btk-dependent pathways in B cells. Further examination of the PDK1 pathway will be achieved through gene targeting approaches to ablate PDK1, Akt and the FOXO proteins. In addition, we will determine the extent to which Btk, PDK1 and Akt contribute to the splenomegaly/lymphadenopathy and altered B cell differentiation observed in mice lacking Pten in B cells and thus subject to the effects of elevated and sustained PIP3,4,5. Through the generation and characterization of these mice we will learn how PI3K directs B cell differentiation, and provide insight into how such regulation can go awry in the case of B cell transformation.